MiR-411-5p was decreased in the HFHCD-induced MASLD rat model

To explore the expression of miR-411-5p in MASLD, we fed SD rats a HFHCD to establish the MASLD model and recorded their body weight regularly (Fig. 1, a–b). The serum ALT, AST, TC and LDL-c levels of rats in the MASLD group were higher than those of the control group, while the HDL-c levels were lower than those of the control group (Fig. 1, c–g). Moreover, the liver weight index and triglyceride (TG) levels of MASLD rats also increased (Fig. 1, h–i). Importantly, liver histopathology revealed numerous instances of hepatocellular steatosis and lipid deposition in the livers of MASLD rats (Fig. 1, j).

Fig. 1

MiR-411-5p was decreased in the HFHCD-induced MASLD rat model. a Schematic diagram showing the establishment of the MASLD rat model. b Rat body weight curve during the modeling period. c-i Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in rats at the end of modeling. (N = 5/group). j H&E staining and Oil Red O staining of rat liver tissue sections and quantitative analysis. (N = 5/group). k Fluorescence in situ hybridization for miR-411-5p in rat liver tissue sections. l Quantification of fluorescence in situ hybridization signal intensity for miR-411-5p in rat liver tissue sections. (N = 5/group). m Relative expression levels of miR-411-5p in rat liver determined by RT-qPCR analysis. (N = 5/group). n Spearman rank correlation analysis of relative expression levels of miR-411-5p and ALT, AST, liver weight index or TG levels, respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Next, we detected significant reduction of miR-411-5p in the liver of MASLD rats (Fig. 1, k-l). Same findings were validated by RT-qPCR. Particularly, hepatic miR-411-5p in the liver of MASLD rats was about 40% lower than that of normal rats (Fig. 1, m). In addition, we found that miR-411-5p was negatively correlated with serum ALT content and AST content, and was also negatively correlated with liver triglyceride levels (Fig. 1, n). Taken together, miR-411-5p was decreased during MASLD, which was related to liver lesions and damage in MASLD.

MiR-411-5p was decreased in the CDAHFD-induced MASLD mouse model

To further clarify the miR-411-5p expression during MASLD, we also established a MASLD mouse model using a CDAHFD and recorded their body weight weekly (Fig. 2, a–b). The serum ALT, AST and LDL-c levels of mice in the MASLD group were higher than those of the control group, while the TC and HDL-c levels were lower than those of the control group (Fig. 2, c–g). Moreover, MASLD mice had a greater liver index and liver TG levels than the control mice (Fig. 2, h–i). Liver histopathology showed numerous steatotic hepatocytes and lipid deposits in the livers of the MASLD group of mice (Fig. 2, j).

Fig. 2

MiR-411-5p was decreased in the CDAHFD-induced MASLD mouse model. a Schematic diagram showing the establishment of the MASLD mouse model. b Mouse body weight curve during the modeling period. c-i Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in mice at the end of modeling. (N = 5/group). j H&E staining and Oil Red O staining of mouse liver tissue sections and quantitative analysis. (N = 5/group). k Fluorescence in situ hybridization for miR-411-5p in mouse liver tissue sections. l Quantification of fluorescence in situ hybridization signal intensity for miR-411-5p in mouse liver tissue sections. (N = 5/group). m Relative expression levels of miR-411-5p in mouse liver determined by RT-qPCR analysis. (N = 5/group). n Spearman rank correlation analysis of relative expression levels of miR-411-5p and ALT, AST, liver weight index or TG levels, respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Importantly, compared with normal mice, miR-411-5p expression was reduced in the livers of MASLD mice (Fig. 2, k-m). In addition, miR-411-5p level was negatively correlated with serum ALT, AST, liver weight index, and liver triglyceride levels in MASLD mice (Fig. 2, n). These data further confirmed that miR-411-5p is decreased in MASLD models.

MiR-411-5p alleviated lipid deposition in hepatocytes

Albumin (ALB) is a hepatocyte-specific marker. In fluorescence in situ hybridization analysis combined with immunofluorescence experiments showed that ALB-labeled cells have a signal of miR-411-5p (Fig. 3, a). We further found that the expression of miR-411-5p in rat liver parenchymal cells in the MASLD group was significantly lower than that in the Normal group, but there was no difference in the expression of miR-411-5p in rat liver non-parenchymal cells in the MASLD group and the Normal group (Fig. 3, b). Similar findings were found in mice (Fig. 3, c). In addition, after PA treatment, the expression of miR-411-5p in HepG2 cells decreased, while the expression of miR-411-5p in macrophages and hepatic stellate cells did not change (Fig. 3, d). The miR-411-5p level in the PA + miR-411-5p group was hundreds of times higher than that in the PA + NC group, indicating that we successfully up-regulated the expression of miR-411-5p (Fig. 3, e). Subsequent BODIPY staining analysis revealed that compared with the PA + NC group, the lipid deposition in the PA + miR-411-5p group was significantly reduced (Fig. 3, f-g).

Fig. 3

MiR-411-5p alleviated lipid deposition in hepatocytes. a Fluorescence in situ hybridization combined with immunofluorescence experiment for miR-411-5p (red) and ALB (green) in rat or mouse liver tissue sections (DAPI, blue). b-c Relative miR-411-5p levels in parenchymal cells and non-parenchymal cells of rat or mouse liver determined by RT-qPCR analysis. (N = 5/group, normalized to U6). d Relative miR-411-5p levels in HepG2 cells, macrophages and LX2 cells determined by RT-qPCR analysis. (N = 5/group, normalized to U6). e Relative expression levels of miR-411-5p in PA-induced steatosis hepatocytes determined by RT-qPCR analysis. (N = 6/group, normalized to U6). f BODIPY staining of PA-induced steatosis hepatocytes. g Quantification of BODIPY staining signal intensity in PA-induced steatosis hepatocytes. (N = 3/group). h–k Relative expression levels of lipid uptake-related genes (CD36, FATP1, FATP2), lipid synthesis-related genes (ACC1, FASN, SREBP1c), lipid oxidation-related genes (ACOX1, CPT1A, PPARα) and lipid transport-related genes (APOB, APOC, APOE) in PA-induced steatosis hepatocytes determined by RT-qPCR analysis. (N = 6/group, normalized to β-actin). l Protein levels of ACC1, FASN, and SREBP1 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group, normalized to Tubulin protein). m TC levels in PA-induced steatosis hepatocytes determined by ELISA analysis. (N = 6/group). *P < 0.05, **P < 0.01, ***P < 0.001

Next, we examined expression changes in the genes involved in lipid uptake, synthesis, oxidation, and transport. MiR-411-5p inhibited the expression of CD36, fatty acid transport protein 2 (FATP2) and apolipoprotein B (APOB), but increased the expression of fatty acid transport protein 1 (FATP1) and apolipoprotein C (APOC) (Fig. 3, h, k). We found that the expression levels of three lipid synthesis-related genes, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and SREBP1c, were all inhibited by miR-411-5p (Fig. 3, i). MiR-411-5p promoted the expression of peroxisome proliferator-activated receptor alpha (PPARα), but had no effect on the expression of acyl-CoA oxidase 1 (ACOX1) or carnitine palmitoyltransferase 1 (CPT1) (Fig. 3, j). Besides, compared with the PA + NC group, the protein levels of SREBP1, ACC1 and FASN in the PA + miR-411-5p group were decreased (Fig. 3, l). MiR-411-5p also reduced the content of TC in PA-HepG2 cells (Fig. 3, m).

EIF4G2 was a direct target of miR-411-5p

After demonstrating that miR-411-5p could reduce lipid deposition, we further explored the underlying mechanism. Using three prediction databases (TargetScan, miRDB, and miRTarBase), we found that four genes (EIF4G2, CDH2, KPNA2, SF3B3) were most likely directly regulated by miR-411-5p (Fig. 4, a). With the upregulation of miR-411-5p, only the EIF4G2 mRNA expression was inhibited (Fig. 4, b-e). In addition, the EIF4G2 protein expression was reduced by the miR-411-5p mimic (Fig. 4, f). In the livers of rats and mice, the expression of EIF4G2 in the MASLD group was higher than that in the control group (Fig. 4, g-h). Sequence alignment analysis suggested that there is a binding site between miR-411-5p and the EIF4G2 3′ untranslated region (Fig. 4, i). Importantly, miR-411-5p mimic effectively reduced the luciferase activity of EIF4G2 in the WT group, but had no effect in the MUT group (Fig. 4, j-k). Therefore, EIF4G2 is the gene directly regulated by miR-411-5p.

Fig. 4

EIF4G2 was a direct target of miR-411-5p. a Three miRNA-target gene prediction databases (miRDB, TargetScan, miRTarBase) were used to identify potential miR-411-5p targets. b-e Relative expression levels of potential miR-411-5p targets (EIF4G2, CDH2, KPNA2, SF3B3) determined by RT-qPCR analysis. (N = 6/group). f Protein levels of EIF4G2 determined by western blot analysis. (N = 6/group). g Protein levels of EIF4G2 in rat liver determined by western blot analysis. (N = 5/group). h Protein levels of EIF4G2 in mouse liver determined by western blot analysis. (N = 5/group). i The binding of the EIF4G2 3′UTR with miR-411-5p. j Dual-luciferase reporter assay to evaluate the effect of miR-411-5p mimic or inhibitor on wild-type EIF4G2 3′UTR. (N = 3/group). k Dual-luciferase reporter assay to evaluate the effect of miR-411-5p mimic or inhibitor on mutant EIF4G2 3′UTR. (N = 3/group). *P < 0.05, **P < 0.01, ***P < 0.001

Down-regulation of EIF4G2 alleviated lipid deposition in hepatocytes

RT-qPCR analysis revealed that compared with the PA + NC group, the EIF4G2 mRNA levels in the PA + siEIF4G2 group decreased by more than 80% (Fig. 5, a). Moreover, the EIF4G2 protein level decreased by more than 70% under the influence of siEIF4G2 (Fig. 5, b). BODIPY staining revealed that compared with the PA + NC group, the degree of lipid deposition in the PA + siEIF4G2 group was significantly weaker (Fig. 5, c-d). In addition, the expressions of ACC1, FASN, and SREBP1 were significantly reduced under the influence of siEIF4G2 (Fig. 5, e). Knockdown of EIF4G2 also reduced the content of TC in PA-HepG2 cells (Fig. 5, f). These results demonstrate that the downregulation of EIF4G2 alleviates lipid deposition in PA-induced steatosis in hepatocytes.

Fig. 5

Down-regulation of EIF4G2 alleviated lipid deposition in hepatocytes. a Relative expression levels of EIF4G2 in PA-induced steatosis hepatocytes determined by RT-qPCR analysis. (N = 6/group). b Protein levels of EIF4G2 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). c-d BODIPY staining of PA-induced steatosis hepatocytes and quantification of BODIPY staining signal intensity. (N = 3/group). e Protein levels of ACC1, FASN, and SREBP1 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). f TC levels in PA-induced steatosis hepatocytes determined by ELISA analysis. (N = 6/group). *P < 0.05, **P < 0.01, ***P < 0.001

Down-regulation of EIF4G2 alleviated lipid deposition in MASLD mice

As in vitro experiments revealed that the downregulation of EIF4G2 improved lipid deposition, we further investigated the effect of EIF4G2 in MASLD mice. We injected the virus-based AAV8 vectors carrying shEif4g2 or shNC into MASLD mice and recorded their body weight weekly (Fig. 6, a–b). Under the influence of AAV8 carrying shEif4g2, serum ALT, AST and LDL-c levels decreased significantly in the shEif4g2 group of MASLD mice compared with the shNC group (Fig. 6, c–g). In addition, compared with MASLD mice in the shNC group, the liver weight index and liver TG levels of MASLD mice in the shEif4g2 group decreased (Fig. 6, h–i). Importantly, H&E staining revealed that the number of fat vacuoles in the liver tissue of MASLD mice in the shEif4g2 group was reduced compared to that in the shNC group (Fig. 6, j, l). Oil Red O staining also showed that lipid deposition in liver tissue has improved in the shEif4g2 group (Fig. 6, k, m). Subsequent western blotting analysis showed that the EIF4G2 protein level in the livers of MASLD mice in the shEif4g2 group was reduced under the influence of AAV8 carrying shEif4g2 (Fig. 6, n). Downregulation of EIF4G2 also resulted in decreased expression of ACC1, FASN, and SREBP1 in the livers of MASLD mice (Fig. 6, o). Overall, the downregulation of EIF4G2 expression effectively reduced fatty acid synthesis and lipid deposition in the livers of MASLD mice.

Fig. 6

Down-regulation of EIF4G2 alleviated lipid deposition in MASLD mice. a Schematic diagram showing the intervention of MASLD with AAV8 carrying shEif4g2 or negative control in the MASLD mouse model. b Mouse body weight curve during the intervention period. c-i Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in mice at the end of intervention. (N = 5/group). j-m H&E staining and Oil Red O staining of mouse liver tissue sections and quantitative analysis. (N = 5/group). n Protein levels of EIF4G2 in mouse liver tissue determined by western blot analysis. (N = 5/group). o Protein levels of ACC1, FASN, and SREBP1 in mouse liver tissue determined by western blot analysis. (N = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001

MiR-411-5p targeted EIF4G2 to reduce FOXO3 expression to alleviate lipid deposition

Immunofluorescence staining of the liver tissue showed that the fluorescence signal of FOXO3 in the livers of MASLD mice in the shEif4g2 group was weaker than that in the livers of MASLD mice in the shNC group (Fig. 7, a). Moreover, compared with MASLD mice in the shNC group, the FOXO3 protein levels in the shEif4g2 group were reduced (Fig. 7, b). In addition, compared with the control group, the fluorescence signal and protein levels of FOXO3 in the miR-411-5p and siEIF4G2 groups were decreased (Fig. 7, c-d).

Fig. 7

MiR-411-5p targeted EIF4G2 to reduce FOXO3 expression to alleviate lipid deposition. a Expression of FOXO3 in mouse liver tissue determined by immunofluorescence. b Protein levels of FOXO3 in mouse liver tissue determined by western blot analysis. (N = 5/group). c Expression of FOXO3 in PA-induced steatosis hepatocytes determined by immunofluorescence. d Protein levels of FOXO3 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). e Protein levels of FOXO3 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). f-g BODIPY staining of PA-induced steatosis hepatocytes and quantification of BODIPY staining signal intensity. (N = 3/group). h Protein levels of ACC1, FASN, and SREBP1 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). i TC levels in PA-induced steatosis hepatocytes determined by ELISA analysis. (N = 6/group). *P < 0.05, **P < 0.01, ***P < 0.001

Next, we used FOXO3-siRNA to successfully downregulate the FOXO3 expression in steatosis hepatocytes (Fig. 7, e). BODIPY staining revealed that down-regulation of FOXO3 effectively reduces lipid deposition in hepatocytes (Fig. 7, f-g). Besides, the levels of ACC1, FASN, and SREBP1 were significantly reduced when FOXO3 was downregulated (Fig. 7, h). Knockdown of FOXO3 also reduced the content of TC in PA-HepG2 cells (Fig. 7, i).

MiR-411-5p alleviated lipid deposition in the HFrD-induced MASLD mouse model

We established a MASLD mouse model using a HFrD and injected the agomir to upregulate miR-411-5p, and recorded their body weight regularly (Fig. 8, a–b). RT-qPCR results showed that miR-411-5p was successfully up-regulated in the liver of MASLD mice (Fig. 8, c). Under the influence miR-411-5p, serum ALT, AST, TC and LDL-c levels decreased significantly in the MASLD mice (Fig. 8, d–h). In addition, compared with MASLD mice in the NC group, the liver weight index and liver TG levels of MASLD mice in the miR-411-5p group decreased (Fig. 8, i–j). Importantly, H&E staining and Oil Red O staining showed that lipid deposition in liver tissue has improved in the miR-411-5p group (Fig. 8, k). Western blotting analysis showed that the EIF4G2 and FOXO3 protein level in the livers of MASLD mice in the miR-411-5p group was reduced (Fig. 8, l). Upregulation of miR-411-5p also resulted in decreased expression of ACC1, FASN, and SREBP1 in the livers of MASLD mice (Fig. 8, m).

Fig. 8

MiR-411-5p alleviated lipid deposition in the HFrD-induced MASLD mouse model. a Schematic diagram showing the establishment of the MASLD and intervention. b Mouse body weight curve during the intervention period. c Relative expression levels of miR-411-5p in mouse liver determined by RT-qPCR analysis. (N = 5/group). d-j Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in mice at the end of intervention. (N = 5/group). k H&E staining and Oil Red O staining of mouse liver tissue sections and quantitative analysis. (N = 5/group). l Protein levels of EIF4G2 or FOXO3 in mouse liver tissue determined by western blot analysis. (N = 5/group). m Protein levels of ACC1, FASN, and SREBP1 in mouse liver tissue determined by western blot analysis. (N = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001

MiR-411-5p inhibited EIF4G2/FOXO3 axis to reduce fatty acid synthesis in mouse primary hepatocytes

After PA treatment, the expression of miR-411-5p in mouse primary hepatocytes decreased (Fig. 9, a). RT-qPCR results showed that miR-411-5p mimic significantly increased the expression of miR-411-5p in PA-treated mouse primary hepatocytes (Fig. 9, b). With increased expression of miR-411-5p, the EIF4G2 and FOXO3 protein levels in PA-treated mouse primary hepatocytes were reduced (Fig. 9, c). In addition, the protein levels of SREBP1, ACC1 and FASN in the PA + miR-411-5p group were significantly lower than those in the PA + NC group (Fig. 9, d). MiR-411-5p also reduced the content of TC in PA-treated mouse primary hepatocytes (Fig. 9, e).

Fig. 9

MiR-411-5p inhibited EIF4G2/FOXO3 axis to reduce fatty acid synthesis in mouse primary hepatocytes. a-b Relative miR-411-5p levels in mouse primary hepatocytes determined by RT-qPCR analysis. (N = 5/group, normalized to U6). c Protein levels of EIF4G2 or FOXO3 in mouse primary hepatocytes determined by western blot analysis. (N = 5/group). d Protein levels of ACC1, FASN, and SREBP1 in mouse primary hepatocytes determined by western blot analysis. (N = 5/group). e TC levels in mouse primary hepatocytes determined by ELISA analysis. (N = 5/group). f The expressions of miR-411-5p reduces during the progression from normal liver to MASLD. The reduction of miR-411-5p leads to an increase in the expression of EIF4G2, which upregulates FOXO3 and promotes lipid synthesis in hepatocytes. *P < 0.05, **P < 0.01, ***P < 0.001